Amplification samples of 5 μL were loaded into glass capillary tubes (1.02 mm o.d., 0.56 mm i.d.) and sealed. The tubes were cleaned with optical-grade methanol and then loaded into the carousel of a fluorescence temperature cycler described elsewhere (23). For continuous monitoring of a single sample, the carousel was positioned at maximal fluorescence and signals acquired every 200 ms. For multiple tubes, signals were obtained once each cycle by sequentially positioning the carousel at each tube for 100 ms.
Figure 1 illustrates the three different fluorescence techniques used for continuous monitoring of DNA amplification. Figures 2, 3 and 4) demonstrate the application of these techniques for initial template quantification by fluorescence monitoring once each cycle. In Figure 2, the fluorescence of the dsDNA-specific dye SYBR Green I is followed. A 107–108 range of initial template concentration can be discerned. When the data are normalized as the percent maximal fluorescence of each tube, 100 initial copies are clearly separated from 10 copies. However, the difference between 1 and 10 copies is marginal, and no difference is observed between 0 and 1 average copies per tube. Nonspecific detection of undesired products after many cycles is a limitation of fluorescence monitoring of dsDNA.
Specific fluorescence monitoring can be obtained with sequence-specific fluorescent probes. In Figure 3, amplification is monitored using a dual-labeled hydrolysis probe. The fluorescence signal is expressed as a ratio of fluorescein to rhodamine f luorescence. Signal generation with 5′-exonuclease probes is dependent not only on DNA synthesis, but requires hybridization and hydrolysis between the fluorophores of the dual-labeled probe. Hydrolysis reduces quenching of fluorescein, and the fluorescence ratio of fluorescein-to-rhodamine emission increases. Whereas the fluorescence from dsDNA-specific dyes plateaus with excess cycling, the signal from hydrolysis probes continues to increase after many cycles. Even though no net product is being synthesized, probe hybridization and hydrolysis continue to occur. In contrast to dsDNA dyes, no fluorescence signal is generated in the absence of template.
In Figure 4, amplification is monitored using adjacent hybridization probes and is expressed as a ratio of Cy5 to fluorescein fluorescence. One of the probes is labeled 3′ with fluorescein, and the other probe is labeled 5′ with Cy5. When hybridized to accumulating product, the probes are separated by a 1-bp gap, and the Cy5-to-fluorescein fluorescence ratio increases. The change in fluorescence ratio during hybridization is largely due to an increase in Cy5 fluorescence from resonance energy transfer (data not shown). In contrast to hydrolysis probes, the fluorescence signal of hybridization probes tends to decrease with excessive cycling. No fluorescence signal is generated in the absence of template.
Sequence-specific probes have an even greater dynamic range for template quantification than do dsDNA dyes. As the template copy number decreases below 103, signal intensity decreases because specific amplification efficiency decreases, but low copy numbers can still be quantified because the negative control signal is stable (Figures 3 and 4). Although multiple samples would need to be run to confirm Poisson statistics, it appears that these specific techniques can discriminate a single initial template copy from negative controls (compare 0 and 1 average initial template copies in Figures 3 and 4).
With each technique, the fluorescence response is not strictly proportional to the amount of specific product. With SYBR Green I, two factors nonspecific amplification of alternative templates results in fluorescence unrelated to specific product, particularly after many cycles (Figure 2). In addition, when the fluorescence of purified DNA standards is measured, the response is only linear to 10–20 ng of DNA per 5-μL reaction under the conditions used (data not shown). Higher concentrations of DNA show proportionally less fluorescence, presumably because the amount of SYBR Green I becomes limiting. Higher concentrations of SYBR Green I than those used here (<1:7000 dilution) inhibit amplification (data not shown). With hydrolysis probes, the fluorescence signal continues to increase after the plateau phase has been reached (Figure 3). With hybridization probes, the fluorescence decreases during the plateau phase (Figure 4). Despite the nonlinearity of these fluorescence techniques, they are very useful for absolute quantification of initial template copy number when fluorescence is measured for each amplification cycle (Figures 2–4).